Project summary Eukaryotic genomes contain arrays of tandemly repeated non-coding sequences that we currently know little about?satellite DNAs. Typically found near centromeres, telomeres and on Y chromosomes, satellite DNAs can comprise over 50% of some eukaryotic genomes. They are known to change rapidly in sequence and genomic location, which can cause genetic incompatibilities between closely related species. The misregulation of satellite DNA can have serious consequences for genomic stability and cancer formation. Despite being a ubiquitous part of genomes and having important functional consequences, we know little about satellite DNA. The lack of genetic, genomic and molecular tools to study tandemly repeated sequences has stymied progress towards understanding satellite DNA evolution and function. For example, satellite DNAs are particularly challenging to sequence and assemble. Recent developments in next-generation sequencing technologies circumvent some of these problems. This proposal integrates genomic, molecular and cytological methods to study the evolutionary and functional genomics of satellite DNA in Drosophila genomes. The PI has developed new genomic and cytological methods to study the evolutionary dynamics, genomic structure and expression of satellite DNA with unprecedented resolution. The PI will use these methods to study changes in satellite DNA sequence, abundance and organization over evolutionary time and to determine the evolutionary forces responsible for these changes. This proposal aims to develop comprehensive models of satellite DNA evolution that take into consideration different types of natural selection based on the functional aspects of satellite DNAs. Little is currently known of satellite DNA function: the precise genetic manipulation of satellite DNAs with site-specific approaches had not been possible in the past due to a lack of unique target sites. The new genomic methods developed in this proposal provide an opportunity to discover unique sites flanking satellite DNAs that may serve as targets for genome editing techniques. The proposal will create precise genomic deletions of satellite DNA in Drosophila melanogaster to test specific hypotheses about the regulation, fitness effects and selfish genetic behavior of satellite DNA. This proposal will also use new molecular genetic techniques to manipulate the expression of satellite DNAs in the germline to ask questions about their functions in chromosome segregation and chromatin organization. These experiments will have broad implications not only for genome evolution, but also for understanding the regulation of satellite DNA in cancer and aging.